Transcytosis vehicles and enchancers for drug delivery

ABSTRACT

Transcytosis of a physiologically-active agent that exerts its action following passage across endothelia, epithelia or mesothelia containing the GP60 receptor is enhanced by formulation with or conjugation to a transcytosis enhancer or vehicle selected from albumin and fragments thereof, anti-GP60 antibody and fragments thereof, GP60 peptide fragments, and PDI (protein disulphide isomerase) and fragments thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. national phase application corresponding toInternational Patent Application No. PCT/GB96/02326, filed Sep. 20, 1996(pending).

This application claim benefit to provisional application Ser. No.60/004,097 Sep. 21, 1995.

FIELD OF THE INVENTION

The invention relates to drug delivery. In particular, the inventionrelates to transcytosis vehicles and enhancers capable of delivering andenhancing passage of drugs across endothelia, epithelia and mesotheliacontaining the GP60 receptor.

BACKGROUND OF THE INVENTION

For most therapeutic drugs administered by intra-arterial or intravenousroutes the intended site of molecular activity lies outside thevasculature. For drugs administered via the airways, the intended siteof activity normally is beyond the first cellular barrier of alveolar,bronchiolar or tracheal epithelia. In both cases, there is anendothelial or epithelial barrier which must be crossed before the drugcan mediate its effect.

For small lipophilic drugs, there appears to be a paracellular routebetween the tight junctions of the barrier cells. However, forhydrophilic drugs and larger macromolecular active agents, such aspeptides, proteins, genes or anti-sense nucleotides, the only routeacross the barrier is through the cells. This poses a particular problemfor drugs administered intravenously which have exceedingly shorthalf-lives due to rapid degradation or first pass clearance by theliver. In order to maintain therapeutic levels in balance with suchexcretion and degradation, large doses or infusions are often necessary.Thus, there is clearly a need in the art for more rapid mechanisms fordelivering drugs across cellular barriers.

There have been numerous reports of specific receptors which mediateendocytotic events, where a ligand binds to the receptor and is theninternalized, complexed to the receptor, by a process similar topinocytosis. This involves invagination of the cell membrane in theregion of the ligand receptor complex and then release of the ligandinto the cell by a process which is not fully understood. Numerousendocytotic receptor systems have been reported including LDL, insulin,epidermal growth factor, insulin-like growth factor and tPA-PAI-I(hybrid molecule).

Transcytosis entails invagination and vesicle formation around a ligandreceptor complex, followed by transcytotic passage with release by areverse invagination process at the basolateral membrane. Monoclonalantibodies to the transferrin receptor have been conjugated with toxins,so that they can undergo transcytosis, across blood-brain endothelia.However, there is a continuing need in the art for agents capable ofdelivering or enhancing passage of drugs by receptor-mediatedtranscytosis across cellular barriers other than blood-brain endothelia,such as endothelia of the vasculature, alveolar epithelia, andperitoneal mesothelia.

The GP60 receptor, also referred to as albondin, is one of severalalbumin-binding proteins reported in the literature (Schnitzer and Oh,J. Biol. Chem. 269(8):6072-6082 (1994)). Others include SPARC (serumprotein, acidic, rich in cysteine), oesteonectin or basement membraneprotein 40, GP30, GP18 and GP60. SPARC and oesteonectin areextra-cellular proteins. GP60 shares some homology with SPARC asdetermined using anti-SPARC antibodies (Schnitzer and Oh, Am. J.Physiol. 263:H1872-H1879 (1992)).

GP18 and GP30 are membrane glycoproteins found in a variety of celltypes but are particularly prevalent in the macrophage (Schnitzer et al,J. Biol. Chem. 267: 24544-24553 (1992)). GP18 and GP30 are the so-called“scavenger receptors” responsible for mediating removal of oxidized,glycated or adduced forms of albumin by endocytosis and are thusbelieved to play a role in albumin catabolism for a wide variety oforgans (Schnitzer and Bravo, J. Biol. Chem. 268(10):7562-7570 (1993)).

In contrast to GP18 and GP30, the GP60 receptor has found to beexpressed exclusively in continuous endothelia of the vasculature(Schnitzer, Am. J. Physiol. 262:H246-H254 (1992)), in alveolar epithelia(Kim et al, Am. J. Resp. and Crit. Care Med. 151:A190, (1994) andinferentially in peritoneal mesothelia (Gotloib and Shostak, KidneyInternational. 47:1274-1284 (1995)). GP60 is particularly abundant inthe microvessel endothelia and is, interestingly, absent from theblood-brain barrier, where little albumin flux is observed (Rousseaux etal, Methods in Enzymology 121:163 (1986)). It has been shown thatpolyclonal antibodies to endothelial GP60 also bind alveolar epithelialGP60 (Kim et al, supra). The GP60 receptor has been implicated inreceptor-mediated transcytosis of albumin across epithelia andendothelial cell barriers (Kim et al, supra; Tirrupathi et al, MolecularBiology of the Cell 4 (Supp):338a, Abstract No. 1964 (1993)).

The GP60 amino acid sequence is known in the art (Yamauchi et al,Biochem. Biophys. Res. Comm. 146:1485 (1987)).

SUMMARY OF THE INVENTION

The present invention provides transcytosis vehicles and enhancerscapable of transporting physiologically-active agents across epithelia,endothelia and mesothelia containing the GP60 receptor. The GP60receptor has been implicated in receptor-mediated transcytosis ofalbumin across cell barriers. By means of the invention, GP60receptor-mediated transcytosis can be exploited for the transport of notonly albumin, but also physiologically-active agents which do notnaturally pass through epithelia, endothelia and mesothelia via the GP60system.

Transcytosis vehicles and enhancers of the invention include albumin,albumin fragments, anti-GP60 polyclonal and monoclonal antibodies,anti-GP60 polyclonal and monoclonal antibody fragments, and GP60 peptidefragments. Further, they include PDI (protein disulphide isomerase) andfragments thereof (any subsequent reference to GP60 fragments may beinterpreted as referring also to PDI fragments). A common factor may bea CGMC motif found in PDI and at least the T₁₋₄₄ fragment of GP60. Ifthe transcytosis vehicle or enhancer is a GP60 peptide fragment, it ispreferably co-administered with other transcytosis vehicles or enhancersof the present invention such as albumin or an albumin fragment.Suitable albumin fragments of 14, 20 and 32 kDa can be generated bycleavage at methionine residues using cyanogen bromide and can befurther reduced in size by reduction of disulfide bridges. Anti-GP60polyclonal and monoclonal antibody fragments useful as transcytosisvehicles and enhancers according to the present invention include Fab,Fab′, F(ab′)₂, and Fv fragments. Preferred GP60 peptide fragmentsinclude the T3118 peptide which corresponds to the N-terminal 18 aminoacids of the GP60 protein.

In accordance with the invention, when the above compounds areconjugated to a physiologically-active agent, they are referred toherein as “transcytosis vehicles”. When co-administered with but notconjugated to a physiologically-active agent, the above compounds arereferred to herein as “transcytosis enhancers”. In preferredembodiments, the transcytosis vehicles and enhancers of the presentinvention are useful for delivering or enhancing passage ofphysiologically-active agents across endothelia of the vasculature,alveolar epithelia and peritoneal mesothelia.

DETAILED DESCRIPTION OF THE INVENTION

As its name indicates, the GP60 protein has been reported in the art ashaving a molecular weight of about 60 kDa. After a more carefulanalysis, it has been discovered that the “true” molecular weight forthis protein is more probably about 57 kDa. This discrepancy inmolecular weight is thought to be due to differences in proteinpreparation and gel conditions. However, to be consistent with the art,this protein is referred to herein (with the exception of Example 1below) as the GP60 receptor.

It has been discovered that GP60 receptor-mediated transcytosis can beexploited for the transport of not only albumin, but also for a vastnumber of therapeutically-important physiologically-active agents whichdo not naturally pass through epithelia, endothelia and mesothelia viathe GP60 system. Thus, the present invention provides an improved methodfor transporting physiologically-active e.g. those having relativelyhigh molecular weights, e.g. 50, 100, 150 kDa or more, across thecellular barriers of the endothelia of the vasculature, alveolar,bronchiolar, and tracheal epithelia, and the peritoneal mesothelia.Transcytosis vehicles and enhancers capable of delivering or enhancingpassage of physiologically-active agents across GP60-containingendothelia, epithelia and mesothelia include albumin, albumin fragments,anti-GP60 polyclonal and monoclonal antibodies, anti-GP60 polyclonal andmonoclonal antibody fragments, and GP60 peptide fragments. If thetranscytosis vehicle or enhancer is a GP60 peptide fragment, it willpreferably be co-administered with other transcytosis vehicles orenhancers of the present invention such as albumin or an albuminfragment.

Mammalian albumin is well known in the art and readily available.Preferably, the albumin used will be from the same mammalian species asthe patient. For example, if the patient is human, human serum albuminwill preferably be used as the transcytosis vehicle or enhancer.Similarly, if the patient is equine or bovine, equine or bovine serumalbumin is preferably used, respectively.

Methods for generating albumin fragments are well known in the art. Forexample, cleavage of albumin at methionine residues by cyanogen bromideyields three particularly suitable peptides of 14, 20 and 32 kDa whichcan be further reduced in size by reduction of the disulfide bridges, topeptides ranging in size from 3.3-20 kDa. Alternatively, proteasedigestion can be used to generate albumin peptide fragments.

Whether any particular albumin fragment is useful as a transcytosisvehicle or enhancer according to the present invention can be determinedaccording to the routine screening assay described below. As indicatedin the Examples below, it has now been demonstrated that both bovine andhuman serum albumin, acting as transcytosis enhancers, stimulate uptakeof a physiologically-active agent 2.5-4 fold over the control.

Anti-GP60 polyclonal and monoclonal antibodies can be generated from theGP60 receptor purified from endothelia, epithelia or mesothelia. Asdiscussed above, endothelial, epithelial and mesothelial cells whichexpress the GP60 receptor include endothelia of the vasculature(including capillary endothelia (Ghinea et al, J. Cell Biol. 107:231-239(1988)); arterial endothelia (Silflinger-Birnboim et al, J. CellularPhysiology 149:575-584 (1991); aortic and vein endothelia (Schnitzer andOh, Am. J. Physiol. (1992), supra); epithelia of alveolar tissue (Kim etal, supra); and mesothelia of the peritoneum (Gotloib and Shostak,supra). GP60 can be purified from epithelia, endothelia and mesotheliaaccording to art-known methods (see, for example, Schnitzer and Oh, J.Biol. Chem. (1994), supra) and as described in Example 1 below.

Producing polyclonal antibodies against purified GP60 or a GP60 peptidefragment (such as the T3118 peptide discussed below) can occur in mice,rabbits, or goats according to art-known techniques. In Example 1 below,the GP60 receptor was eluted from preparative SDS-PAGE to immunizerabbits. Approximately 50 μg protein per rabbit was injectedintramuscularly after mixing with equal volume of Freund's completeadjuvant. A second injection was given after two weeks. Rabbits werebled at 4 to 6 weeks after the second injection, and the immune responsewas tested. The antiserum IgG was then purified using a ProteinA-Sepharose column.

Monoclonal antibody preparation can also occur according to knowntechniques (Goding, J. Immunol. Methods 39:285 (1980); Oi andHerzenberg, Selected Methods in Cellular Immunology, p. 352, Freeman,San Francisco, 1979)). For example, Balb/c mice are injectedintraperitoneally with 50-150 μg of GP60 or a GP60 peptide fragment.Three to five days before the fusion, positive mice receive a boosterinjection of antigen (50-150 μg of GP60 or GP60 fragment), and then 10μg (intravenous and intraperitoneal route) every day until spleenremoval. The spleen cells are fused with Sp2/0-Ag14 myeloma cellsessentially according to St. Groth et al, J. Immunology Methods 35:1-21(1980). Culture supernatants are screened by ELISA using unconjugatedGP60 or GP60 fragment as antigen. Positive cultures are then tested byimmunofluorescence and Western blotting on cDNA-transfected COS-1 cellsas described in Lutz et al, Experimental Cell Research 175:109-124(1988). Hybridomas secreting specific antibodies are cloned twice onsoft agar. Each hybridoma can be adapted in serum-free medium SFRI-4.For ascites fluid production, approximately 2×106 cells are injected inpristine-primed Balb/c mice. Class and subclass determination isperformed using an Isotyping Kit. Both SFRI culture supernatants andascites fluids can be used as monoclonal antibody sources.

As discussed, the anti-GP60 polyclonal and monoclonal antibodies andantibody fragments of the present invention are useful as transcytosisvehicles and enhancers capable of delivering or enhancing passage ofphysiologically-active agents across endothelia, epithelia andmesothelia containing the GP60 receptor. Anti-GP60 antibody fragmentsuseful as transcytosis vehicles or enhancers of the present inventioninclude fragments containing single (Fab) antigen binding domainsproduced by papain digestion; or F(ab′)₂ fragments produced by limitedpepsin digestion (Olsson and Kaplan, Methods in Enzymology 92:3 (1983)).Other suitable fragments include Fab′ and Fv. Whether any particularantibody fragment is useful as a transcytosis vehicle or enhancer can bedetermined according to the routine screening assay described below. InExample 3 below, it is demonstrated that administering anti-GP60polyclonal antibodies at 37° C. results in a 1.6-2 fold increase inuptake of a physiologically-active agent over the level of a pre-immuneserum control.

According to the invention, anti-GP60 antibodies raised in animals otherthan humans such as mice and rats are suitable for short-termadministration only (i.e., non-chronic dosage) due to the well-knownadverse immune response to foreign antibodies. However, art-describedmethods can be used to produce human monoclonal antibodies to the GP60receptor, to overcome the problems of administering murine monoclonalsto humans (Olsson and Kaplan supra), thereby rendering the antibodiessuitable for long-term or chronic administration. Moreover, the murineantibodies of the present invention can be “humanized” by chimeric orCDR grafting. The recognition region of the murine antibody is graftedinto the appropriate region of a human antibody, in order to avoid orlimit an adverse immune response in a patient.

GP60 peptide fragments are also useful as transcytosis vehicles andenhancers according to the present invention. Particularly suitable GP60peptide fragments include the first 18 amino acids from the N-terminusof GP60; it has been discovered that this is at least 80% homologous toa stretch of the bovine, membrane-bound thyroid hormone (T3) bindingprotein. Such GP60 peptide fragments can be produced according to anyknown enzymatic or physical technique, including proteolyticdegradation. Alternatively, GP60 peptide fragments can be producedsynthetically. As indicated in Example 5 below, a synthetic N-terminalpeptide (T3118) of GP60 corresponding to the first 18 residues may beproduced by solid-phase synthesis. This peptide, acting as an agonist oftranscytosis, stimulated uptake of human albumin 5-fold over thecontrol.

Methods for conjugating the transcytosis vehicles of the presentinvention to a physiologically-active agent will be readily apparent tothe skilled artisan and include, but are not limited to, glutaraldehydeconjugation involving Schiff base formation; carbodiimide reactionbetween proteins and carboxylic acids; acid anhydride activation ofamine-containing drugs followed by carbodiimide linkage; activation ofprimary amine-containing drugs with3-(2-pyridyldithio)propionate-N-succinimidyl anhydride followed bycoupling to cysteine groups of proteins; coupling of sugar alcohols toproteins utilizing cyanuric chloride; and conjugation between amines andhydroxyl groups via bisperoxidation.

For example, the amino sugar moiety of a physiologically-active agentcan be oxidized by sodium periodate treatment and directly attached tolysine residues on a transcytosis vehicle of the present invention viaSchiff base formation according to the method described in Hurwitz etal, Cancer Res. 35:1175-1181 (1975). Alternatively, aphysiologically-active agent can be linked to a transcytosis vehicle ofthe present invention through carbodiimide-mediated linkage of the aminogroup of the active to carbonyl groups on the vehicle or to anaminoalkyl group according to the method described in Hurwitz et al,Int. J. Cancer 21:747-755 (1978). The physiologically-active agent canalso be linked to a transcytosis vehicle of the present invention bycross-linking the amino sugar of the active and amino groups of thevehicle with glutaraldehyde according to the method described inBelles-Isles et al, Br. J. Cancer 41:841-842 (1980).

Other suitable conjugation sites for conjugating physiologically-activeagents to one of the transcytosis vehicles of the present invention canbe routinely determined empirically. For example, a transcytosis vehicleof the present invention can be labelled with fluorescein or ¹²⁵I eitherbefore or after conjugation to a physiologically-active agent such asinsulin. After conjugation and labelling, a screening assay such as thatdescribed in the Examples below can be used to determine the endothelialcell uptake, the epithelial cell flux, or the mesothelial cell flux ofany candidate vehicle/active conjugate. Such a routine screening assayallows the skilled artisan to determine which transcytosis vehicles ofthe present invention retain the ability to undergo transcytosis afterbeing conjugated at a particular site to a physiologically-active agent.Such an assay is also useful for routine screening of candidate albuminfragments, anti-GP60 antibody fragments and GP60 peptide fragments todetermine which are suitable (for use as transcytosis vehicles andenhancers according to the present invention.

The conjugation of physiologically-active agents to a transcytosisvehicle of the present invention is particularly suited for intravenousdelivery of low molecular weight drugs which otherwise have exceedinglyshort serum half-lives, or of peptide drugs that are rapidly degraded inthe blood stream or removed by first pass excretion in the liver. Ofcourse, where the physiologically-active agent is covalently conjugatedto one of the transcytosis vehicles of the present invention, theresidual activity of the therapeutic agent must be assessed afterconjugation. Techniques for assaying a therapeutic agent's activity arewell established in the art, and many therapeutics have successfullybeen conjugated and retained substantial activity. For example, theliterature describes conjugates between receptor ligands, or fragmentsthereof, and drugs to promote transcytosis across the blood brainbarriers. Fukta et al, Pharm. Research 11(12):1681 (1994), describeconjugation of horse radish peroxidase (HRP) to insulin which enabledHRP to cross the blood, brain barrier. The investigators went on toproduce fragments of insulin which were screened for their ability tobind to the insulin receptor on bovine brain microvessel endothelialcells in culture. Similarly, other transcytosis systems allow thepassage of antibodies linked with active drugs including, among others,antibody-methotrexate targeted to the transferrin receptor (Friden etal, Proc. Natl. Acad. Sci. USA 88:4771 (1991)), and antibody-polylysinetargeted to the epidermal growth factor receptor (Chen et al, FEBS Lett.338:167 (1994)).

By contrast to the transcytosis vehicles, transcytosis enhancers of theinvention are not conjugated to the physiologically-active agent. It hasbeen discovered that co-residence on epithelia, endothelia andmesothelia containing the GP60 receptor of one of the transcytosisenhancers of the present invention and a physiologically-active agent issufficient to enhance uptake and passage of the agent across the cellbarrier. Without wishing to be bound by theory, the transcytosisenhancers of the present invention apparently “trigger” theGP60-mediated transcytosis mechanism, thereby stimulating the enhanceduptake of co-resident macromolecules, including therapeutic agents.

Uptake or passage of physiologically-active agents by or acrossepithelia, endothelia and mesothelia can be induced or enhanced with anyof the transcytosis enhancers of the present invention either alone orin combination. For example, the experiments below demonstrate that,acting as an agonist of transcytosis, the GP60 peptide T3118 enhancedhuman albumin uptake 5-fold over the control. In a further embodiment ofthe present invention, delivery of active agents can be achieved whenone of the transcytosis vehicle conjugates discussed above isadministered together with one or more of the transcytosis enhancers ofthe present invention.

The transcytosis vehicle conjugates and the transcytosis enhancercompositions (including an active agent) of the present invention can beadministered with a pharmaceutically-acceptable carrier or excipient,i.e., pharmaceutically-acceptable organic or inorganic substancessuitable for application which do not deleteriously react with theconjugate or composition. Suitable pharmaceutically-acceptablesubstances include but are not limited to water, salt solutions,alcohol, vegetable oils, polyethylene glycols, gelatin, lactose,amylose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid monoglycerides and diglycerides, petroethralfatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc.The pharmaceutical preparations can be sterilized and, if desired, mixedwith auxiliary agents, e.g., lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, colourings, flavouring and/or aromatic substances, which do notdeleteriously react with the conjugates. For parenteral application,particularly suitable preparations are solutions, preferably oily oraqueous solutions, as well as suspensions, emulsions, or implants,including suppositories. Ampoules are convenient unit dosages. Forenteral application, particularly suitable preparations are tablets,dragees or capsules having a carrier binder such as talc and/or acarbohydrate, the carrier preferably being lactose and/or corn starchand/or potato starch. A syrup, elixir or the like can be used wherein asweetened vehicle is employed. Sustained release compositions can beformulated including those wherein the active component is protectedwith differentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

Administration of a conjugate or composition comprising one or morephysiologically-active agents and one or more of the transcytosisvehicles or enhancers of the present invention can occur according toany art-known technique including injection or via the pulmonaryairways. Injection is particularly suitable for administration to thevasculature and the peritoneum, whereas the pulmonary airways areparticularly suitable for administration to the alveoli. Suitableformulations for pulmonary administration include one or more of thetranscytosis enhancers of the present invention admixed with aphysiologically-active agent. Alternative suitable formulations forpulmonary administration include a transcytosis vehicle conjugated tothe agent. For example, formulations may be made from a nebulizer devicesuch as an Acorn or DeVilbiss jet nebulizer, wherein the agent andtranscytosis enhancer or vehicle are presented as an aqueous solution inthe nebulizer reservoir. Alternatively, in a preferred embodiment forpulmonary administration, the formulation is discharged from a drypowder inhaler (DPI) device. DPI devices are described by Sutton et alin U.S. patent application Ser. No. 08/487,420 and in WO-9609814. Theyrequire spray-drying the formulation into microparticles of 2-5 μm whichare preferred for alveolar penetration.

In particular, a transcytosis enhancer or vehicle of the presentinvention or a mixture thereof, preferably at a concentration of about20% w/v, is used for spray-drying. The preparation to be sprayed maycontain substances other than the transcytosis enhancers or vehicles andsolvent or carrier liquid. For example, the aqueous phase may contain1-20% by weight of water-soluble hydrophilic compounds such as sugarsand polymers as stabilizers, e.g., polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), gelatin, polyglutamic acidand polysaccharides such as starch, dextran, agar, xanthin and the like.Similar aqueous phases can be used as the carrier liquid in which thefinal microsphere product is suspended before use. Emulsifiers may beused (0.1-5% by weight), including most physiologically-acceptableemulsifiers, for instance egg lecithin or soya bean lecithin, orsynthetic lecithins such as saturated synthetic lecithins, for example,dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, ordistearoyl phosphatidylcholine or unsaturated synthetic lecithins, suchas dioleyl phosphatidylcholine or dilinoleyl phosphatidylcholine.Emulsifiers also include surfactants such as free fatty acids, esters offatty acids with polyoxyalkylene compounds, e.g. polyoxypropylene glycoland polyoxyethylene glycol; ethers of fatty alcohols withpolyoxyalkylene glycols; esters of fatty acids with polyoxyalkylatedsorbitan; soaps; glycerol-polyoxyethylene ricinoleate; homo-andcopolymers of polyalkylene glycols; polyethoxylated soya-oil and castoroil as well as hydrogenated derivative; ethers and esters of sucrose orother carbohydrates with fatty acids, fatty alcohols, these beingoptionally polyoxyalkylated; mono-, di- and triglycerides of saturatedor unsaturated fatty acids, glycerides or soya-oil and sucrose.

Additives can be incorporated into the wall of the microspheres tomodify the physical properties such as dispersibility, elasticity andwater permeability. Among the useful additives include compounds whichcan “hydrophobize” the wall in order to decrease water permeability,such as fats, waxes and high molecular weight hydrocarbons. Additiveswhich improve dispersibility of the microspheres in the injectableliquid-carrier are amphipathic compounds such as phospholipids; theyalso increase water permeability and rate of biodegradability. Additiveswhich increase wall elasticity include plasticizers such as isopropylmyristate and the like. The quantity of additives to be incorporated inthe wall is extremely variable and depends on the needs. In someapplications, no additive is used at all; in other cases, amounts ofadditives which may reach about 20% by weight of the wall are possible.

A solution containing one or more transcytosis enhancers or vehicles ofthe present invention and additive, if any, is atomized and spray-driedby any suitable technique which results in discrete microspheres ormicrocapsules of 2 to 5 μm as discussed above. As used herein,“microcapsules” refers to hollow particles enclosing a space, whichspace is filled with a gas or vapour but not with any solid materials.

The atomization forms an aerosol of the transcytosis vehicle or enhancerformulation, for example by forcing the formulation through at least oneorifice under pressure, or by using a centrifugal atomizer in a chamberof warm air or other inert gas. The chamber should be big enough for thelargest ejected drops not to strike the walls before drying. The gas orvapour in the chamber is clean (preferably sterile and pyrogen-free) andnon-toxic when administered to the bloodstream in amounts concomitantwith administration of the microcapsules in use. The rate of evaporationof the liquid from the preparation should be sufficiently high to formhollow microcapsules but not so high as to burst the microcapsules. Therate of evaporation may be controlled by varying the gas flow rate,concentration of transcytosis vehicle or enhancer in the formulation,nature of liquid carrier, feed rate of the solution and, moreimportantly, the temperature of the gas encountered by the aerosol. Forexample, an albumin or albumin fragment concentration of 15-25% inwater, and an inlet gas temperature of at least about 100° C.,preferably at least 110° C., is sufficient to ensure hollowness and thetemperature may be as high as 250° C. without the capsule bursting.About 180-240° C., preferably about 210-230° C. and most preferablyabout 220° C., is optimal. Since the temperature of the gas encounteredby the aerosol will depend also on the rate at which the aerosol isdelivered and on the liquid content of the preparation, the outlettemperature may be monitored to ensure an adequate temperature in thechamber. An outlet temperature of 40-150° C. is suitable. Controllingthe flow rate is useful in controlling other parameters such as thenumber of intact hollow particles.

The microparticles may comprise at least 50%, more preferably 70% or80%, and most preferably 90%, by weight transcytosis enhancer. For usein an inhaler device, the microparticles may be formulated with aconventional excipient such as lactose or glucose. The amount of thephysiologically-active agent will be chosen with regard to its natureand activity, to the mode of administration and other factors known tothose of skill in the art. By way of example, the number of particlesadministered may be such as to deliver 100 mg/day α-1 anti-trypsin, or0.1 mg/day of an active agent such as beclomethasone. Other possiblephysiologically-active agents that can be administered viamicroparticles are given below.

A further embodiment of the present invention is the co-spray-drying ofthe physiologically-active agent with the transcytosis enhancer in orderto facilitate stabilization of the active agent during formulation,packing, and most importantly, during residence on the alveolar lining.In this environment, there can be intense proteolytic activity. In thisor another embodiment, the active agent may be covalently linked to thetranscytosis vehicle via cleavable linkages prior to spray-drying. Thisembodiment represents a method of carrying the active agent all the wayfrom the device to the bloodstream, and possibly to targets within thebody. The formation of particles with optimal aerodynamic size meansthat the “physical” vehicle delivers the active agent to the site ofabsorption. Once deposited upon the alveoli, the “molecular” vehiclethen protects and facilitates passage into the bloodstream via theGP60-mediated transcytosis system and, once in the bloodstream, canfurther enhance circulatory half-life and even direct the active agentto certain sites which are found to contain the GP60 receptor. Suitablelinking technologies are discussed above; further, WO-A-9317713describes esterase-sensitive polyhydroxy acid linkers. Such technology,used in the derivatization of the transcytosis vehicle prior tospray-drying, enables the production of a covalent carrier system fordelivery of active agents to the systemic vasculature. This utilizes thepotential of the transcytosis vehicles to cross the alveoli and to carryactive agents over a prolonged period while protecting potentiallyunstable entities.

Although the physiologically-active agent used in the present inventionmy be imbibed into or otherwise associated with the microparticles aftertheir formulation, it is preferably formulated with the transcytosisvehicle or enhancer. The microparticles may be at least partly coatedwith a hydrophobic or water-insoluble material such as a fatty acid, inorder to delay their rate of dissolution and to protect againsthydroscopic growth.

Methods and equipment for spray-drying and generating themicroparticles, e.g. for use in a dry powder inhaler device aredescribed in more detail in WO-A-9609814 and in U.S. patent applicationSer. No. 08/487,420, the contents of which are incorporated herein byreference.

The optimal proportions of drug to transcytosis enhancer in aformulation for pulmonary delivery can be determined according to anysuitable method. An in vitro optimization of the formulation entailsusing epithelial monolayers of primary human or immortalized humanepithelial cells grown as monolayers on porous filters, as described inthe Examples below. Combinations of drug and enhancer may then beapplied to the upper chamber of a transwell flux system also asdescribed below. Using either labelled tracer or an immunoassay, fluxrates of the drug or gene to the lower layer are determined. The optimalformulation is defined as the one showing maximal rate and extent ofpassage through the restrictive monolayer.

An alternative way of optimizing the formulation entails performing anin vivo determination of lung to blood passage of the drug underinvestigation. There are well-reported studies in rat, pig and sheep(Patton et al, Journal of Controlled Release 28:79 (1994), Folkesson etal, Acta. Physiol. Scand. 147:73 (1993); Schreier et al, Pharm. Res.11:1056 (1994)); these studies describe methods of instilling oraerosolizing drug formulations into the trachea and bronchioles andassessing the appearance in blood of the drug by immunoassay orpharmacological activity. Optimization would entail a series of animalpreparations using differing proportions of the drug and enhancer, theoptimal formulation being defined by the most beneficial area under thecurve that matched the desired pharmacological profile for the drug. Forinstance, the drug may simply be required to show the maximalbioavailability or alternatively to show a protracted or sustainedrelease profile. For each case, it is likely that there would bediffering requirements for the level of enhancer incorporated in theformulation. For drugs requiring maximal availability, it would bedesirable to utilize the maximal level of enhancer and/or the enhancershowing the highest activating effect upon the GP60 receptor. For drugsrequiring a longer period of presentation across the lung, it would bedesirable to utilize lower levels of enhancer and/or enhancers showinglower activation potential on the transcytosis GP60 receptor.

The “strength” of the enhancer or vehicle can be defined, by the extentto which transcytosis of a given tracer can be enhanced, by the presenceof the GP60 receptor-binding ligand, antibody or mimetic, over the levelof transcytosis in the absence of the ligand. The “strength” of theenhancing agent may be somewhat drug-dependent also. Enhancement ofmarker uptake can vary dependent upon the nature of the marker and thetranscytosis enhancer. Tabulated below is a synopsis of the markers,enhancers, cell system and extent of enhancement over the controlachieved for differing markers cell systems and experimental type.

Abbreviations used:

¹²⁵I-BSA ¹²⁵Iodine-labelled bovine albumin ¹²⁵I-IgG ¹²⁵Iodine-labelledImmunoglobulin G HSA Human albumin BSA Bovine albumin FITC-Insulinfluorescein-labelled insulin GP60 Ab Anti-GP60 polyclonal antibody T3118Synthetic peptide derived from N terminal 18 residues of GP60 FoldMarker Enhancer Cell Type Enhancement ¹²⁵I-BSA GP60 AbBovine/Endothelia/ 1.6 flux ¹²⁵I-BSA GP60 Ab Bovine/Endothelia/ 2.0 fluxanti-BSA BSA Bovine/Endothelia/ 1.5 ¹²⁵I-IgG flux FITC- HSA Human 2.5Insulin Endothelia/flux FITC- BSA Rat Epithelia/flux 4 Insulin ¹²⁵I-BSABSA/T3118 Bovine 5 Endothelia/uptake

By “physiologically-active agent” is intended drugs which includenucleic acid molecules and medicinal peptides and proteins.“Physiologically-active agent” is used interchangeably herein with“drug”, “active”, “active agent” and “therapeutic”. Drugs that wouldbenefit from a more rapid transcytosis across the endothelia andepithelia include Luteinizing hormone (LH), chorionic gonadotropin,atrial peptides, interferon, the various lymphokines such as theinterleukins (I, II, III, IV, V, VI, and VII), and colony-stimulatingfactors.

Other drugs suitable for use in the present invention include: Growthhormone-releasing factor, corticotropin-releasing factor, luteinizinghormone-releasing hormone (LHRH), somatostatin, calcitonin,thyrotropin-releasing hormone, calcitonin gene-related peptide (CGRP),proteins such as enzymes, including transferases, hydrolases,isomerases, proteases, ligases, oxidoreductases, esterases andphosphatases, and various growth and neurotrophic factors, such assomatomedins, epidermal growth factors, urogastrone, nerve growth factor(NGF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophicfactor (BDNF), glial-derived neurotrophic factor (GDNF), epidermalgrowth factor (EGF), fibroblast growth factor (FGF), insulin-like growthfactor, tumour necrosis factor (TNF) and transforming growth factor(TGF). Further drugs include endogenous opioid agonists, such asencephalins and endorphins; hypothalamic hormones, such asgonadoliberin, melanostatin, melonoliberin, somatostatin, thyroliberin,substance P, and neurotensin; adenohypophyseal hormones, such ascorticotropin, lipotropin, melanotropin, lutropin, thyrotropin,prolactin, and somatotropin; neurohypophyseal hormones; calcitrapic(thyroid) hormones, such as parathyrin and calcitonin; thymic factors,such as thymosin, thymopoietin, circulating thymic factor, and thymichumoral factor; pancreatic hormones, such as insulin, glucagon andsomatostatin; gastrointestinal hormones, such as gastrin,cholecystokinin, secretin, gastric inhibitory polypeptide,vasointestinal peptide, and motillin; ovarian hormones, such as relaxin;vasoactive tissue hormones, such as angiotensin and bradykinin; andartificial or pseudo peptides, such as deferoxamine; and LHRH analogssuch as buserelin, deslorelin, gonadorelin, goserelin, histrelin,leuprorelin, nafarelin, or triptorelin.

Having generally described the invention, the same will be more readilyunderstood through reference to the following Examples which areprovided by way of illustration but are not intended to be limiting.

EXAMPLE 1

Growth of Endothelial and Epithelial Monolayers

Bovine pulmonary microvessel endothelial cells (BPMVEC) and (BPAEC)bovine pulmonary artery endothelial cells were isolated and culturedaccording to described methods (Del Vecchio et al, In Vitro. Cell. Dev.Biol. 28A:711-715 (1992)). Endothelial cells were routinely culturedwith DMEM containing 20% FBS. For isolating plasma membranes, theendothelial cells were cultured in 850 cm³ roller bottles. To eachroller bottle, 75 ml culture medium was added. An air-CO₂ mixture wasintroduced. The cells were then transferred to a roller bottle incubatorat 37° C., and were allowed to grow for 10-12 days.

Primary rat alveolar epithelial cells (AEC) were isolated by methodsdescribed in Uhal et al, Am. J. Physiol. 257:C528-C536 (1989). Cellswere cultured in DMEM containing 10% FBS for either 2 or 4 days, atwhich times they exhibited a type II or type I cell-like phenotyperespectively. Phenotype was verified by methods described by Uhal et al,Am. J. Physiol. Suppl. 261:110-117 (1991).

Endothelial Cell Membrane Isolation

Endothelial cells grown in roller bottles were washed 2× with phosphatebuffered saline. The cells were scraped from roller bottles andsuspended in Buffer-A (20 mM HEPES/Tris, 0.15 M NaCl, 0.1 mM PMSF at pH7.4) and washed 2× by centrifuging at 700×g for 10 minutes. The cellsobtained from 6-8 roller bottles were suspended in 75 ml of buffer-A andhomogenized using a Polytron homogenizer for 1 minute at full speed. Thehomogenate was centrifuged at 3000×g for 10 minutes. The supernatant wascollected and centrifuged at 40,000×g for 60 minutes. The pelletobtained was then suspended in buffer-A and recentrifuged at 40,000×gfor 60 minutes. The final membrane pellet was suspended in a smallvolume of buffer-A containing 0.2 mM EDTA and the protein concentrationwas determined (Lowry et al, J. Biol. Chem. 193:265-275 (1951)). Theplasma membrane marker enzyme activities were determined and the samplestored at −70° C. until further use.

Ligand Blotting

Endothelial cell membranes were preincubated with 1 mM PMSF and 0.5 mMEDTA for 20 minutes at 22° C., and then solubilized by mixing with 1.5volume of solubilizing buffer (9M urea, 2% SDS, 2% β-mercaptoethanol,0.1 M Tris, 0.02% bromophenol blue pH 6.8). The mixture was incubated at22° C. for 30 minutes. The solubilized proteins were separated bySDS-PAGE (Laemmli, Nature (London) 227:680-685 (1970)) using a slab-gelelectrophoretic system with 3% acrylamide in the stacking gel and 10%acrylamide in the separating gel. After electrophoresis, the proteinswere transferred to either PVDF or nitrocellulose membrane. The transferwas carried out for 2 hours at 150 volts using 25 mM Tris, 192 mMglycine, and 20% methanol as transfer buffer. The non-specific bindingwas blocked by incubating the membrane with 5 mM CaCl₂ in TBS (20 mMTris, 0.5 M NaCl at pH 7.5) for 10 minutes and then with 0.5% Tween-20in TBS overnight. After this step, the membrane was washed and cut intotwo strips. One strip was incubated with 0.6 mg/ml globulin-free BSA inTBS containing 1.5% gelatin for 2 hours and the other strip wasincubated without BSA. The strips were washed and incubated withanti-bovine BSA for 60 minutes in TBS containing 1.5% gelatin. Themembranes were then washed 2× and incubated with second antibody (goatanti-rabbit IgG) conjugated with alkaline phosphatase. The protein bandswere localized after adding 5-bromo-4-chloro-3-indolylphosphate andnitroblue tetrazolium salt.

Protein Purification

BPMVEC membranes were used to isolate a 57 kDa albumin-binding protein.The ligand blotting was carried out to assess the presence of thisprotein in each step. BPMVEC membranes (100 mg) were preincubated with 1mM PMSF and 0.5 mM EDTA for 30 minutes at 22° C. The membranes weresolubilized using a final concentration of 2.5% sodium cholate and 4 Murea, at 4° C. for 3 hours, with gentle stirring. The proteinconcentration was adjusted to 4 mg/ml during solubilization. After thistreatment, the suspension was centrifuged at 100,000×g for 60 minutes.The supernatant was collected and dialyzed against 5 mM HEPES/Tris (pH7.2). More than 80% of membrane proteins were recovered in thesupernatant. The dialysed suspension was concentrated by 60% ethanolprecipitation at 4° C. The ethanol precipitate was collected bycentrifugation at 10,000×g for 30 minutes at 4° C. and suspended inBuffer-A. This precipitate was solubilized with 2.5% Triton X-100overnight at 4° C. with gentle stirring. The suspension was centrifugedat 100,000×g for 60 minutes. The supernatant was collected and dialysedagainst 4 l of 50 mM Tris-HCl, 0.2 mM EDTA, 0.15% Triton X-100 and 0.1mM PMSF, pH 8.0 (Buffer-B). The dialysed extract was applied on aDEAE-52 column (10×13 cm). The column was previously equilibrated withBuffer-B. The column was washed with 50 ml of Buffer-B after applyingthe sample. The bound proteins were eluted from the column with 80 ml of0-500 mM linear NaCl gradient in Buffer-B at a flow rate of 15 ml/hr.The fractions from individual peaks were pooled separately andconcentrated by 50% acetone precipitation. The acetone precipitate wasused for ligand blotting. Only peak-I showed albumin-binding activity.The proteins present in peak-I were further separated by usingpreparative SDS-PAGE (16 cm×16 cm, 3 mm thick slab-gel), and a 57 kDaprotein eluted from the gel was used for further studies.

Antibody Production and Purification

The 57 kDa albumin-binding protein eluted from preparative SDS-PAGE wasused to immunize rabbits. Approximately 50 μg protein (per rabbit) wasinjected intramuscularly after mixing with equal volume of Freund'scomplete adjuvant. A second injection was given after two weeks. Rabbitswere bled at 4 to 6 weeks after the second injection and the immuneresponse was checked. The preimmune serum IgG and the antiserum IgG werepurified using protein A-sepharose column.

Immunoblotting

Endothelial cell membranes were subjected to SDS-PAGE (Laemmli, supra) ,and electrophoretically transferred to nitrocellulose or PVDF membrane.Non-specific binding was blocked with 3% gelatin in TBS for 5 hours at22° C. The membrane was washed 2× with 0.5% Tween-20 in TBS andincubated with antiserum diluted in TBS containing 1% gelatin. Theincubation was carried out for 4-6 hours, washed 2×, and then incubatedfor 60 minutes with the second antibody (goat anti-rabbit IgG coupled toalkaline phosphatase). After incubation, the membranes were washed 2×and the protein bands were localized as described under “LigandBlotting”. Molecular weights of the proteins were determined using knownmarker proteins.

Monolayer Binding Studies

BPMVEC were seeded (3×105 cells/well) in six well Corning tissue cultureplates and grown to confluence. The monolayers were washed 2× withserum-free medium (20 mM HEPE/DMEM pH 7.4) and incubated with serum-freemedium for 15-20 hours in a tissue culture incubator. After thisincubation, the monolayers were washed 2× with binding buffer (20 mMHEPES/Tris HBSS pH 7.4) and the binding was initiated by adding 1 ml of1 μM ¹²⁵I-BSA in binding buffer. The incubation was carried out at 4° C.for 60 minutes. The binding was terminated by washing the monolayer 3×with the binding buffer. The radioactivity associated with the monolayerwas determined after lysing the cells with 1 N NaOH (Tiruppathi et al,Am. J. Physiol. (Lung. Cell. Mol. Physiol.) L595-L601 (1992)).Non-specific binding was determined by the inclusion of unlabelled BSA(40 mg/ml) during the binding procedure. The test components, preimmuneserum-IgG and the anti-57 kDa-IgG were preincubated for 30 minutes withthe monolayer prior to the addition of ¹²⁵I-BSA.

Trans-cellular Flux Experiments

Transendothelial ¹²⁵I-albumin flux rates in cultured endothelialmonoloyers were used to assess transendothelial albumin transport. Thesystem used for this study has previously been described (Cooper et al,J. Appl. Physiol. 62:1076-1083 (1987); Garcia, et al, J. Cell. Physiol.128:96-104 (1986); Del Vecchio, et al, Vitro. Cell. Dev. Biol.28A:711-715 (1992) and Siflinger-Birnboirn et al, J. Cell. Physiol.132:111-117 (1987)). The system measures the transendothelial movementof tracer macromolecules in the absence of hydrostatic and oncoticpressure gradients. It consists of luminal and albuminal compartmentsseparated compartments separated by a polycarbonate microporous filter(0.8 μm pore diameter). BPMVEC were seeded at 105 cells/filter and grownfor 3-4 days to attain confluency. Both compartments contained the samemedium (20 mM HEPES-DMEM, pH 7.4) at volumes of 600 ml and 25 ml,respectively. The luminal compartment was fitted with a Styrofoam outerring, and “floated” in the abluminal medium so that fluid levelsremained equal after repeated samplings from the abluminal compartment.The abluminal compartment was stirred continuously and the entire systemwas kept at 37° C. by a thermostatically regulated water bath.Transendothelial clearance of ¹²⁵I-albumin was determined as the volumeof luminal chamber radioactivity cleared into the abluminal chamber. Thechange in volume over time provided the ¹²⁵I-albumin clearance rate inμl/min as determined by weighted least-squares non-linear regressionanalysis (BMDP Statistical Software, Berkeley, Calif.).

At the beginning of the experiment, the luminal compartment was floatedin the abluminal medium, and filled with medium containing about 6μCi/ml ¹²⁵I-albumin. Albuminal samples, 400 μl, were collected at 10minute intervals for up to 60 minutes and the radioactivity was measuredusing a gamma counter. At the end of the experiment, free ¹²⁵I in theluminal and abluminal compartments was determined using 12% TCAprecipitation and the transendothelial ¹²⁵I-albumin flux rates werecorrected for free ¹²⁵I.

The day before the experiment, the BPMVEC monolayers were washed 2× with20 mM HEPES-DMEM pH 7.4 (serum-free medium) and incubated at 37° C. incell culture incubator with serum-free medium for 12-15 hours. Afterthis incubation period, the test components (preimmune serum-IgG and theanti-57 kDA-IgG) were diluted in serum-free medium and incubated withthe monolayers for the desired periods. These monolayers were then usedfor transendothelial albumin transport measurement.

Trans-epithelial flux rates were measured with slight modification tothe method described for endothelial cells. Flux rates were determinedon primary AEC or the A549 human lung carcinoma cell line cultured asdescribed on Transwell filters (Costar) (Evans et al, Exper. Cell Res.18:375-387 (1989)). Monolayer integrity is defined by transepithelialelectrical resistance being greater than 500 ohms/cm₂. Filters withintact monolayers were placed in a 24 well culture plate containing 1 mlserum-free DMEM per well (abluminal chamber). The luminal chamber wasfilled with 200 μl serum-free DMEM containing the tracer molecule ofinterest (FITC-Insulin). The fluid levels in the two compartments werethe same, eliminating hydrostatic pressure. The filter system waspreincubated (30 mins) and then maintained at 37° C. in a CO₂ incubatorthroughout the flux experiment. At one and two hours, 300 μl sampleswere withdrawn from the abluminal chamber and immediately replaced withserum-free DMEM. The fluorescence of the transcytosed material wasrecorded on a plate reader, and the ratio of bound vs. free FITCdetermined by gel filtration chromatography of the abluminal samples.

Actin Filament Distribution

The actin filament distribution and cytoskeletal changes in endothelialmonolayers grown on the filters were studied under the conditionsidentical to those used for the measurement of ¹²⁵I-albumin clearancerates. After the required pretreatment period with the test components,the monolayers on the filter were fixed in 10% buffered formalin(Pallescences Inc., Warrington, Pa.), permeabilized with 1% Nonidet P40(Sigma), and stained with rhodamine phalloidin (Molecular Probes, Inc.,Eugene, Oreg.) as described by Phillips and Tsan, J. Histochem.Cytochem. 36:551-554 (1988). The intact filters containing themonolayers were removed from the wells and mounted on coverslips,covered with a 1:1 solution of glycerine in phosphate-buffered saline,and then covered with a round coverslip and sealed. The slides wereanalyzed using a Nikon Lab Diaphot fluorescent microscope (NiKon Inc.,Melville, N.Y.) and photographed using TRI X Pan 400 ASA Kodak film).

Identification of Albumin-Binding Proteins

Plasma membranes were first isolated from BPMVEC by differentialcentrifugation and the albumin-binding proteins present in this membranefraction were identified using ligand blotting (see above). A simplemethod was developed, to identify native albumin-binding proteins inendothelial cell membranes. The membrane proteins were separated usingSDS-PAGE and then transferred to PVDF or nitrocellulose. Non-specificbinding was blocked by incubating the membrane strips with Tween-20, andthen treated with globulin-free monomeric native BSA. The BSA-bindingregions were identified using polyclonal antibody raised against nativeBSA. In the absence of exposure of the membrane strip to native BSA, theanti-BSA recognized only a 67 kDa polypeptide, indicating the presenceof a significant amount of BSA bound to endothelial cell membranes.However, when the strip was treated with BSA, the anti-BSA antibodyreacted with 3 additional polypeptides (110 kDa, 57 kDa and 18 kDa). Ofthese polypeptides, the antibody reacted most intensely with 57 kDa,indicating the 57 kDa polypeptide to be the major native albumin-bindingprotein. Total endothelial cell membrane fractions (100,000×gparticulate fraction from BPMVEC and BPAEC) were also prepared and usedfor ligand blotting. These particulate fractions also showed a primaryinteraction of BSA with the 57 kDa polypeptide.

Isolation of the 57 kDa Albumin-Binding Protein

Since binding of native albumin was seen primarily with the 57 kDaprotein, a method was developed for the isolation of this protein fromBPMVEC membranes. Ligand blotting was employed to assess the presence ofthis protein during purification. BPMVEC membranes were initiallysolubilized with 2.5% sodium cholate and 4M urea, and the extract wasdialyzed and concentrated by 60% ethanol precipitation. This precipitatewas re-extracted with Triton x-100 (see above). The Triton x-100solubilized extract was chromatographed on the DEAE column, and thebound proteins were eluted with linear gradient (0-500 mM NaCl). Theproteins were eluted as 3 peaks. The fractions from each peak werepooled and screened for albumin-binding using the ligand blotting assay.Only one peak (I) showed albumin-binding with the 57 kDa protein region.

SDS electrophoresis was conducted, using proteins from native BPMVECmembrane and DEAE column peak I after staining with Coomassie brilliantblue R-250. The presence of 57 kDa protein corresponding toalbumin-binding was observed with ligand blotting in both nativemembranes as well as in DEAE peak I. SDS-PAGE was also performed undernon-reducing conditions (in absence of βME), and the albumin-binding wasobserved only with 57 kDa region, suggesting the absence of sulfide linkin this protein. This protein was further purified using preparativeSDS-PAGE, and the protein eluted from gel was used for the antibodypreparation.

Immunoblotting

BPMVEC and BPAEC membrane proteins were separated by using SDS-PAGE andtransferred to nitrocellulose strips. The strips were immunoblotted withthe 57 kDa antiserum. The preimmune serum did not recognize any proteinsfrom BPMVEC and BPAEC membranes. The antiserum recognized two majorproteins (57 kDa and 36 kDa) and one minor protein (43 kDa) in bothmembrane preparations. The particulate fractions from BPMVEC and BPAECwere also used for immunoblotting. The antibody recognized only thesethree proteins in the particulate fractions. This suggests that thealbumin-binding protein was purified to an apparent homogeneity.

To study the proposed structural relationship between the endothelialmembrane-associated and secreted (SPARC) albumin-binding proteins,immunoblotting of BPMVEC membranes was carried out with the antibodiesraised against purified bovine SPARC. The antiserum raised againstpurified bovine SPARC recognized 67 kDa, 61 kDa, 57 kDa, 43 kDa and 36kDa polypeptides in BPMVEC membranes. The anti-SPARC-NH2 terminalpeptide antiserum reacted strongly with a 36 kDa polypeptide and weaklywith a 43 kDa polypeptide. This suggests that scavenger receptors arequite different from native albumin receptors.

Effect of Anti-57 kDa-IgG on Binding of ¹²⁵I-BSA to BPMVEC Monolayers

Preimmune serum-IgG and the anti-57 kDa-IgG were affinity-purified usingProtein-A Sepharose column. The influence of IgG fractions on binding of¹²⁵I-BSA to BPMVEC monolayers at 4° C. was investigated: non-specificbinding ranged from 40-50%. The preimmune serum-IgG did notsignificantly affect the specific binding of ¹²⁵I-BSA to the BPMVECmonolayers. In contrast, the anti-57 kDa-IgG reduced the specificbinding of ¹²⁵I-BSA to BPMVEC monolayers in a dose-dependent manner. Thereduction was maximum (40-50%) at 200 μg/ml concentration in anti-57kDa-IgG, and remained unchanged up to 1000 μg/ml.

These results demonstrate that the antibody developed against the 57 kDaprotein does not fully recognize the albumin-binding domain in thereceptor, or that the native albumin may interact with other bindingsites on endothelial cell surface.

Activation of Transendothelial Albumin Flux by Anti-57 kDa-IRG in theAbsence of Endothelial Cell Shape Change

To study the effects of the anti-57 kDa-IgG on transendothelialtransport of albumin, the transendothelial ¹²⁵I-BSA clearance rates inBPMVEC monolayers was measured. The monolayers were preincubated withpreimmune serum-IgG and anti-57 kDa-IgG for 15 minutes, 30 minutes and60 minutes, and then the transendothelial ¹²⁵I-BSA clearance rates weremeasured up to 60 minutes. The anti-57 kDa-IgG-induced increase inpermeability was time-dependent. A 30-minute period of preincubation ofanti-57 kDa-IgG resulted in a 2-fold increase in ¹²⁵I-BSA clearance rateover preimmune IgG. No significant increase in permeability was seenwith 15 min. preincubation, and a 40-50% change was noticed when anti-57kDa-IgG was pre-incubated with the monolayer up to 60 min. The preimmuneserum-IgG had no influence on transendothelial albumin transport at allpreincubation periods tested. The anti-57 kDa-IgG effect on thepermeability of ¹²⁵I-albumin reverted at 4° C.

The shape change of endothelial cells after treating with preimmuneserum-IgG and anti-57 kDa-IgG was studied, using a technique describedpreviously (Phillips and Tsan, supra; Siflinger-Birnboim et al, LabInvest. 67:24-30 (1992)). BPMVEC grown on nucleopore filters werepreincubated with preimmune serum-IgG and anti-57 kDa IgG for 30 min.,and the monolayers were stained with rhodamine phalloidin (see above).No cell “rounding” or formation of interendothelial gaps was observed ineither case.

These results suggest that anti-57kD albumin-binding protein antibodyactivates albumin transport. There is another possibility, i.e. thatthis antibody may non-specifically increase the pericellular transportof albumin, by widening the interendothelial junctional gaps. Todelineate this, the effect of anti-receptor IgG and preimmune serum IgGon endothelial cell morphology was studied. Pretreatment of BPMVECmonolayers with either preimmune serum-IgG or anti-receptor-IgG had noinfluence on interendothelial junctional gaps. This antibody to the 57kDa albumin-binding protein may activate the transcytosis of albumin.The permeability increasing effect of this antibody did not occur at 4°C., supporting the conclusion that the antibody activated albumintranscytosis via formation of vesicles, which have been shown to betemperature-sensitive (Lo et al, J. Cell. Physiol. 151:63-70 (1992)).

EXAMPLE 2

Antibodies Raised Against GP60

Antibody raised against GP60 from endothelial cells was used to probeepithelial membrane extracts as described in Example 1. The anti-GP60antibodies recognized a 60 kDa protein found in the epithelial extracts.This clearly shows that an immunologically-related protein is present inthis system.

Epithelial and endothelial cells were grown as monolayers, as describedin Example 1, to produce confluent monolayers showing the appropriatereactivity to solute flux. Anti-GP60 antibody (200-500 μg/ml) wasincubated with the monolayers at 4° C. to bind antibody to the receptor,in the absence of metabolic activity that might result ininternalization of the GP60. Binding of anti-GP60 antibody under theseconditions resulted in a 80-90% decrease in ¹²⁵I-BSA binding by theendothelial monolayers. The epithelial monolayers were further incubatedwith a second antibody to the primary rabbit anti-GP60 antibody, tocross-link the receptors. Both monolayers were washed and then incubatedwith ¹²⁵I-BSA for the epithelial cells or ¹²⁵I anti-BSA immunoglobulinfor the endothelial monolayers at 37° C., to allow internalization ofthe receptor-antibody complex and co-transcytosis of the ¹²⁵I-labelledtracer. Incubation with anti-GP60 antibody resulted in a 1.6-2 foldincrease in uptake over the level of a pre-immune serum control. Thus,binding the GP60 receptor by an anti-GP60 antibody results in activationof the transcytosis mechanism, thereby enhancing uptake of amacromolecule in the vicinity of the invaginating membrane.

EXAMPLE 3

Use of Albumin with Macromolecules

Endothelial monolayers were incubated at 4° C. in the presence of BSA,to initiate the binding of BSA to GP60 but to prevent theinternalization of the ligand receptor complex. After extensive washingto remove unbound BSA, the cells were incubated with ¹²⁵I-labelledanti-BSA immunoglobulin at 37° C., as the macromolecular tracer.Pre-treatment with BSA enhanced transcytosis of the immunoglobulintracer by 1.5 fold over the control cells pre-incubated with unlabelledanti-BSA immunoglobulin. Further, when the cells incubated at 37° C.were washed and immediately taken through the same protocol, nomacromolecular flux was observed; this shows that, once internalized,the GP60 receptor is unavailable for ligand binding. Thus, large (150kDa) molecules can be co-transcytosed in concert with HSA using the GP60system.

EXAMPLE 4

Use of Albumin with Peptides

Human and rat epithelial monolayers were grown to confluence, asdescribed in Example 1. The cells were then incubated with FITC-insulin(1 mg/ml) or FITC-insulin and BSA (each 1 mg/ml) at 37° C. in thetranscellular flux system described above. For human and rat epithelialmonolayers, there was a 2.5 or 4 fold increase in FITC-insulin flux overthe control of FITC-insulin alone. Thus, albumin also stimulatesco-transcytosis of small molecular weight peptides across epithelialcells containing the GP60 receptor.

EXAMPLE 5

Use of N-Terminal Peptide 1-18 of GP60

A synthetic N-terminal peptide (T3118) of GP60 corresponding to thefirst 18 residues was produced by solid-phase peptide synthesis. Thesequence (SEQ ID No. 1) shows at least 80% homology with the bovine,membrane-bound thyroid hormone (T3)-binding protein (Yamauchi et al,Biochem. Biophys. Res. Comm. 146:1485 (1987)). It has 97% homology withPDI.

Antibodies were raised in rabbits against T3118, and used to probeendothelial membrane extracts, to determine cross-reactivity withproteins recognized by anti-GP60 antibodies as described below. BPMVECmembrane proteins (100 μg) were separated on SDS-PAGE and transferred tonitroulose membrane strips. Non-specific binding was blocked with 5%non-fat dry milk in Tris-buffered saline. The antisera were diluted inblocking solution, incubated for 4-5 hrs at 4° C., washed and treatedwith goat-anti-rabbit-IgG conjugated with alkaline phosphatase. Theprotein bands were identified using known molecular weight markerproteins. The anti-T3118 antibodies showed only reactivity towards theGP60 protein and not towards the SPARC peptides recognized by theanti-GP60 antibody.

The T3118 peptide was then used in an endothelial uptake experiment todetermine if it would act as an antagonist of albumin recognition anduptake. Endothelial monolayers were incubated at 4° C. in the presenceof ¹²⁵I-BSA or ¹²⁵I-BSA plus the T3118 peptide. After incubation, thecells were washed extensively, lysed and counted for tracer uptake.Surprisingly, rather than acting as an antagonist, the T3118 peptideactually stimulated uptake of albumin 5-fold over the albumin alonecontrol. The enhancement was saturable at a concentration of 500 μm ofT3118 peptide. These data suggest that the T3118 peptide, acting as anagonist, may induce a conformational change in albumin, which enhancesrecognition by GP60, or is the signal for uptake by the endothelialcells.

It will be appreciated by those skilled in the art that the inventioncan be performed within a wide range of equivalent parameters ofcomposition, concentrations, modes of administration, and conditionswithout departing from the spirit or scope of the invention or anyembodiment thereof.

1 1 18 PRT Unknown Description of Unknown Organism GP60 peptide 1 LysPro Asp Glu Glu Asp His Val Leu Val Leu Val Lys Gly Asn Phe 1 5 10 15Asp Val

What is claimed is:
 1. A composition comprising, or conjugate of, aphysiologically-active agent that exerts its action following passageacross endothelia, epithelia or mesothelia containing the GP60 receptor,and a transcytosis enhancer or vehicle selected from albumin andfragments thereof, anti-GP60 antibody and fragments thereof, GP60peptide fragments, and PDI (protein disulphide isomerase) and fragmentsthereof; wherein said composition or conjugate is a dry powder suitablefor inhalation.
 2. The composition or conjugate according to claim 1,wherein the transcytosis enhancer or vehicle includes the CGMC motif. 3.The composition or conjugate according to claim 1, wherein thetranscytosis enhancer or vehicle comprises albumin or an albuminfragment.
 4. The composition or conjugate according to claim 1, whereinthe transcytosis enhancer or vehicle comprises anti-GP60 antibody or ananti-GP60 antibody fragment.
 5. The composition or conjugate accordingto claim 1, wherein the transcytosis enhancer or vehicle comprises aGP60 peptide fragment.
 6. The composition or conjugate according toclaim 1, wherein the transcytosis vehicle comprises albumin or analbumin fragment in combination with a GP60 peptide fragment.
 7. Thecomposition or conjugate according to claim 5 or claim 6, wherein theGP60 peptide fragment is or comprises SEQ ID No.
 1. 8. The compositionor conjugate according to claim 1, wherein the physiologically-activeagent is selected from the group consisting of Luteinizing hormone (LH),chorionic gonadotropin, atrial peptides, interferon, lymphokines I,lymphokine II, lymphokine III, lymphokine IV, lymphokine V, lymphokineVI, lymphokine VII, a colony-stimulating factor, growthhormone-releasing factor, corticotropin-releasing factor, luteinizinghormone-releasing hormone (LHRH), somatostatin, calcitonin,thyrotropin-releasing hormone, calcitonin gene-related peptide (CGRP),transferases, hydrolases, isomerases, proteases, ligases,oxidoreductases, esterases, phosphatases, nerve growth factor (NGF),ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor(BDNF), glial-derived neurotrophic factor (GDNF), epidermal growthfactor (EGF), fibroblast growth factor (FGF), insulin-like growthfactor, tumour necrosis factor (TNF), transforming growth factor (TGF),encephalins, endorphins, gonadoliberin, melanostatin, melonoliberin,somatostatin, thyroliberin, substance P, neurotensin, corticotropin,lipotropin, melanotropin, lutropin, thyrotropin, prolactin,somatotropin, neurohypophyseal hormones, parathyrin, calcitonin,thymosin, thymopoietin, circulating thymic factor, thymic humoralfactor, insulin, glucagon, somatostatin, gastrin, cholecystokinin,secretin, gastric inhibitory polypeptide, vasointestinal peptide,motillin, relaxin, angiotensin, bradykinin, somatomedins, epidermalgrowth factors, urogastrone, deferoxamine, buserelin, deslorelin,gonadorelin, goserelin, histrelin, leuprorelin, nafarelin, andtriptorelin.
 9. In a method for administering a physiologically-activeagent to a mammal, the improvement comprising: administering said activeagent either conjugated to or in admixture with a transcytosis vehicleor enhancer as defined in claim 1, wherein said transcytosis vehicle orenhancer delivers or enhances passage of said physiologically-activeagent across epithelia, endothelia, or mesothelia containing the GP60receptor.
 10. The method of claim 9, wherein said physiologically-activeagent is selected from the group consisting of Luteinizing hormone (LH),chorionic gonadotropin, atrial peptides, interferon, lymphokine I,lymphokine II, lymphokine III, lymphokine IV, lymphokine V, lymphokineVI, lymphokine VII, a colony-stimulating factor, growthhormone-releasing factor, corticotropin-releasing factor, luteinizinghormone-releasing factor (LHRH), somatostatin, calcitonin,thyrotropin-releasing hormone, calcitonin gene-related peptide (CGRP),transferases, hydrolases, isomerases, proteases, ligases,oxidoreductases, esterases, phosphatases, nerve growth factor (NGF),ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor(BDNF), glial-derived neurotrophic factor (GDNF), epidermal growthfactor (EGF), fibroblast growth factor (FGF), insulin-like growthfactor, tumor necrosis factor (TNF), transforming growth factor (TGF),encephalins, endorphins, gonadoliberin, melanostatin, melonoliberin,somatostatin, thyroliberin, substance P, neurostensin, corticotropin,lipotropin, melanotropin, lutropin, thyrotropin, prolactin,somatotropin, neurohypophyseal hormones, parathyrin, calcitonin,thymosin, thymopoietin, circulating thymic factor, thymic humoralfactor, insulin, glucagon, somatostatin, gastrin, cholecystokinin,secretin, gastric inhibitory polypeptide, vasointestinal peptide,motillin, relaxin, angiotensin, bradykinin, somatomedins, epidermalgrowth factors, urogastrone, deferoxamine, buserelin, deslorelin,gonadorelin, goserelin, histrelin leuprorelin, nafarelin, andtriptorelin.
 11. The method of claim 9, wherein saidphysiologically-active agent is conjugated to said transcytosis vehicleby a method selected from the group consisting of glutaraldehydeconjugation using Schiff base formation, carbodiimide reaction betweenproteins and carboxylic acids, acid anhydride activation of aminecontaining drugs followed by carbodiimide linkage, activation of primaryamine containing drugs with3-(2-pyridyldithio)proprionate-N-succinimidyl anhydride followed bycoupling to cysteine groups of proteins, coupling of sugar alcohols toproteins utilizing cyanuric chloride, and conjugation between amines andhydroxyl groups via bisperoxidation.
 12. The method of claim 9, whereinsaid mammal is human.
 13. The composition or conjugate, according toclaim 1, which further comprises an excipient.
 14. The composition orconjugate, according to claim 1, which comprises microparticles of theactive agent which are from 2 to 5 μm in size.
 15. An inhaler devicewhich comprises a composition comprising, or conjugate of, aphysiologically-active agent that exerts its action following passageacross endothelia, epithelia or mesothelia containing the GP60 receptor,and a transcytosis enhancer or vehicle selected from albumin andfragments thereof, anti-GP60 antibody and fragments thereof, GP60peptide fragments, and PDI (protein disulphide isomerase) and fragmentsthereof; wherein said composition or conjugate is a dry powder suitablefor inhalation.